Ka. Westerman et P. Leboulch, REVERSIBLE IMMORTALIZATION OF MAMMALIAN-CELLS MEDIATED BY RETROVIRAL TRANSFER AND SITE-SPECIFIC RECOMBINATION, Proceedings of the National Academy of Sciences of the United Statesof America, 93(17), 1996, pp. 8971-8976
A procedure of reversible immortalization of primary cells was devised
by retrovirus-mediated transfer of an oncogene that could be subseque
ntly excised by site-specific recombination. This study focused on the
early stages of immortalization: global induction of proliferation an
d life span extension of cell populations. Comparative analysis of Cre
/LoxP and FLP/FRT recombination in this system indicated that only Cre
/LoxP operates efficiently in primary cells. Pure populations of cells
in which the oncogene is permanently excised were obtained, following
differential selection of the cells, Cells reverted to their preimmor
talized state, as indicated by changes in growth characteristics and p
53 levels, and their fate conformed to the telomere hypothesis of repl
icative cell senescence. By permitting temporary and controlled expans
ion of primary cell populations without retaining the transferred onco
gene, this strategy may facilitate gene therapy manipulations of cells
unresponsive to exogenous growth factors and make practical gene targ
eting by homologous recombination in somatic cells, The combination of
retroviral transfer and site-specific recombination should also exten
d gene expression studies to situations previously inaccessible to exp
erimentation.